Bottom Line:
ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex.Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation.ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF.

fig2: ZO-1 down-regulation reduces endothelial cell–cell tension. (A and B) Cells were transfected with siRNAs and, after 2 d, with a VE-cadherin–based FRET tension sensor containing (TS) or lacking (Tminus) the β-catenin binding site. FRET activity was then imaged by gain of donor fluorescence after acceptor bleaching from confluent monolayers. (A) FRET efficiency maps and images taken from venus fluorescent protein before bleaching. (B) Images were quantified by calculating the FRET efficiencies at cell–cell contacts. The values were then normalized to the FRET efficiency obtained with the tail-minus construct, which does not sense tension and hence provides the FRET signals that can maximally be expected (shown are means ± 1 SD [error bars]; n = 12). (C–E) Cells expressing GFP–α-catenin were plated and transfected with siRNAs as in A. The cells were then analyzed by ablating single cells (marked with an asterisk) within the monolayer with a laser and recording the movement of cell–cell contacts in the GFP channel for 1 min. The images in C show overlays of frames taken before ablation in red, after 30 s in green, and after 45 s in blue (see also Videos 1 and 2). The increase in the surface area of the ablated cells and the contraction of the neighboring cells were then analyzed (D and E, shown are means ± 1 SD [error bars]; n = 11). Bars, 20 µm.

Mentions:
We next asked whether these changes resulted in altered tensile force on the junctional complex. We used a FRET biosensor based on VE-cadherin that carries an elastic force sensor within the cytoplasmic domain between the p120 and the β-catenin binding sites (Grashoff et al., 2010; Conway et al., 2013). Fig. 2 (A and B) shows that the sensor was sensitive to the presence of the β-catenin binding site, which indicates that the probe responded to forces that act on VE-cadherin. Depletion of ZO-1 also led to increased FRET efficiency, indicating that the tensile force acting on VE-cadherin was diminished (Fig. 2, A and B). Therefore, reduced ZO-1 expression stimulated a reduction in the tension on VE-cadherin, which suggests that ZO-1 regulates tension on adherens junctions.

fig2: ZO-1 down-regulation reduces endothelial cell–cell tension. (A and B) Cells were transfected with siRNAs and, after 2 d, with a VE-cadherin–based FRET tension sensor containing (TS) or lacking (Tminus) the β-catenin binding site. FRET activity was then imaged by gain of donor fluorescence after acceptor bleaching from confluent monolayers. (A) FRET efficiency maps and images taken from venus fluorescent protein before bleaching. (B) Images were quantified by calculating the FRET efficiencies at cell–cell contacts. The values were then normalized to the FRET efficiency obtained with the tail-minus construct, which does not sense tension and hence provides the FRET signals that can maximally be expected (shown are means ± 1 SD [error bars]; n = 12). (C–E) Cells expressing GFP–α-catenin were plated and transfected with siRNAs as in A. The cells were then analyzed by ablating single cells (marked with an asterisk) within the monolayer with a laser and recording the movement of cell–cell contacts in the GFP channel for 1 min. The images in C show overlays of frames taken before ablation in red, after 30 s in green, and after 45 s in blue (see also Videos 1 and 2). The increase in the surface area of the ablated cells and the contraction of the neighboring cells were then analyzed (D and E, shown are means ± 1 SD [error bars]; n = 11). Bars, 20 µm.

Mentions:
We next asked whether these changes resulted in altered tensile force on the junctional complex. We used a FRET biosensor based on VE-cadherin that carries an elastic force sensor within the cytoplasmic domain between the p120 and the β-catenin binding sites (Grashoff et al., 2010; Conway et al., 2013). Fig. 2 (A and B) shows that the sensor was sensitive to the presence of the β-catenin binding site, which indicates that the probe responded to forces that act on VE-cadherin. Depletion of ZO-1 also led to increased FRET efficiency, indicating that the tensile force acting on VE-cadherin was diminished (Fig. 2, A and B). Therefore, reduced ZO-1 expression stimulated a reduction in the tension on VE-cadherin, which suggests that ZO-1 regulates tension on adherens junctions.

Bottom Line:
ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex.Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation.ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF.